Microarrays having a plurality of polymeric molecules spatially distributed over and stably associated with the surface of a substantially planer substrate are becoming an increasingly important tool in molecular biology and related fields. Microarrays of both polypeptide and polynucleotides have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. One area in particular in which microarrays find use is in gene expression analysis.
The current methods of manufacturing microarrays employ a single polynucleotide sequence within each assay element on the microarray. For example, U.S. Pat. No. 5,445,934 discloses a method of on-chip synthesis. In this process, the substrate is derivatized with a chemical species containing a photocleavable protecting group. Selected sites are deprotected by irradiation through a mask. The deprotected sites are then reacted with a DNA monomer containing a photoprotective group. The process of masking, deprotecting and reacting is repeated for each monomer attached until an array of site-specific sequences is achieved. Alternatively, the oligonucleotides may be synthesized directly on modified substrates using ink-jet printing methods disclosed in U.S. Pat. No. 6,015,880. In another method, disclosed in U.S. Pat. No. 6,001,309, the sequences are either presynthesized or isolated and then deposited on the substrate.
A typical method of using microarrays involves contacting nucleotide sequences contained in a fluid with the sequences immobilized on the microarrays under hybridization conditions, and then detecting the hybridization complex. The resultant pattern of hybridized nucleic acids provides information regarding the genetic profile of the sample tested. A widely used method for detecting the hybridization complex in microarrays is by fluorescence. In one method, probes derived from a biological sample are prepared in the presence of nucleotides that have been coupled to a fluorescent label (reporter) molecule so as to create labeled probes, and the labeled probes are then incubated with the microarray so that the probe sequences hybridize to the complementary sequences immobilized on the microarray. A scanner is then used to determine the levels and patterns of fluorescence.
The use of fluorescence detection in microarray analysis is disclosed in U.S. Pat. No. 5,888,742 to Lal et al. for the detection of altered expression of human phospholipid binding protein (PLBP) and in U.S. Pat. No. 5,891,674 to Hillman et al. for the monitoring of the expression level of insulin receptor tyrosine kinase substrate (IRS-p53h), and to identify its genetic variants, mutations and polymorphisms for determining gene function, and in developing and monitoring the activity of therapeutic agents.
A disadvantage of these methods is that since each array element contains one polynucleotide sequence, parallel hybridization assays must be carried out in cases where more than one sequence is used to detect a gene transcript. Consequently, the number of elements that must be arrayed in order to detect a plurality of gene transcripts increases. Further increasing the density of the arrays and microarrays is the need to array control elements in order to detect signal variations and cross hybridization reactions. For example, in microarrays, the signal is affected by the sample to sample variation in printing, the quality and hybridization performance of each array element, and the like. As such, there continues to be interest in the development of new methodologies of manufacturing and utilizing microarrays.